Finally, the gene targets of the differentially expressed miRNAs were predicted, and the functions and signaling pathways of these targets were analyzed using an online database. The isolated exosomes had a bilayer membrane with a diameter of about 100 nm and expressed exosomal markers including CD63, Tsg101, and Flotillin but negatively expressed Calnexin. Both the exosome protein concentration and total protein amount exhibited no significant differences between the two groups. The qPCR assay demonstrated that among the eight miRNAs, the expression levels of miR-942-5p, miR-149-5p, and miR-32-5p in the serum exosomes from the SCAD group were significantly higher than that from the control group. And the three miRNAs for SCAD diagnosis exhibited AUC values of 0.693, 0.702, and 0.691, respectively. GO categories and signaling pathways analysis showed that some of the predictive targets of these miRNAs were involved in the pathophysiology processes of SCAD. In conclusion, our findings suggest that serum exosomal miR-942-5p, miR-149-5p, and miR-32-5p may serve as potential diagnostic biomarkers for SCAD.Exosomal miRNAs are currently being explored as a novel class of biomarkers in cardiovascular diseases. However, few reports have focused on the value of circulating exosomal miRNAs as biomarkers for stable coronary artery disease (SCAD). Here, we aimed to investigate whether miRNAs involved in cardiovascular diseases in circulating exosomes could serve as novel diagnostic biomarkers for SCAD. Firstly, the serum exosomes were isolated and purified by the ExoQuick reagent and identified by transmission electron microscopy, western blot, and nanoparticle tracking analysis. Then, the purified exosomes were quantified by measuring the exosome protein concentration and calculating the total protein amount. Next, eight miRNAs involved in cardiovascular diseases, miR-192-5p, miR-148b-3p, miR-125a-3p, miR-942-5p, miR-149-5p, miR-32-5p, miR-144-3p, and miR-142-5p, were quantified in circulating exosomes from the control group ( Coronary artery disease (CAD) is a heart disease caused by myocardial ischemia or hypoxia because of stenosis or occlusion due to coronary atherosclerosis. Although remarkable achievements have been made in drug and interventional therapies in recent decades, the prevalence and mortality of CAD are still on the rise in many countries , 2. NowaMicroRNAs (miRNAs) are endogenous, single-stranded, and small noncoding RNAs that regulate gene expression at the posttranscriptional level . PreviouExosomes, a kind of endogenous extracellular vesicle with a diameter ranging from 40 to 100 nanometers (nm), contain different bioactive molecules such as RNAs and proteins and play an important role in intercellular communication , 16. miRHowever, few reports have focused on the value of circulating exosomal miRNAs as biomarkers for SCAD. In this study, eight miRNAs, miR-192-5p , miR-148Study subjects were selected from the patients who underwent coronary angiography (CAG) for the evaluation of suspected CAD in the cardiology department of Guangdong Provincial People's Hospital. The inclusion criteria included 40-70 years old, gender unlimited, and diagnosis of SCAD or exclusion of CAD. Those with normal coronary angiograms were included in the control group, and those diagnosed with SCAD were included in the SCAD group. The diagnostic criteria for SCAD included : (1) steTwo milliliters (mL) of peripheral blood samples were obtained from each participant at about 6 a.m. (after an overnight fast) the day after admission. All the blood samples were collected in serum separator tubes containing separation gel and separated into serum fractions by centrifugation at 1000 g for 15 min after standing for half an hour. Serum specimens were frozen at -80°C until used.μL of ExoQuick was added to 250 μL of the centrifuged serum and incubated at 4°C for 30 min. The pellets of exosomes were collected by centrifuging the ExoQuick/serum mixture at 3000 g for 10 min. After that, the isolated exosomes were purified by elution of purification columns. If the purified exosomes were not immediately used for experiments, they should be stored at -80°C and avoid repeated freezing and thawing.The serum samples were thawed at 4°C and then used to isolate exosomes with ExoQuick® ULTRA EV Isolation Kit  according to the manual's instruction. Briefly, the serum samples were centrifuged at 3000 g for 15 min and then centrifuged at 12000 g for 10 min to remove cellular debris. All the centrifugation was performed at 4°C. Subsequently, 67 μL of exosome suspension was loaded onto a formvar-coated copper grid and fixed at least 5 min. Then, the exosomes were negatively stained with 2% uranyl acetate for 5 min. After being air-dried, the grids were visualized with TEM  at 80 kV.The morphology of the isolated exosomes was observed by transmission electron microscopy (TEM). Briefly, 5 μg) were subjected to SDS-PAGE on a 12% polyacrylamide gel and transferred onto nitrocellulose membranes (Merck Millipore). After being blocked with 5% bovine serum albumin (BSA) for 1.5 h at room temperature, the membrane was incubated with anti-CD63 , anti-Tsg101 , anti-Flotillin  antibody, and anti-Calnexin  overnight at 4°C. Then, the membranes were incubated with HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG  for 1 h at 37°C. Finally, the protein bands were visualized by chemiluminescence using the ECL kit (Millipore) and quantified by the ImageJ software.The isolated exosomes were lysed with RIPA lysis buffer , and the protein concentrations were measured by the BCA method. The proteins . The sample chamber was cleaned with particle-free distilled water. Then the exosome samples were diluted 10000× in sterilized PBS. Subsequently, the diluted exosomes were slowly injected into the chamber and quantified using the nanoparticle tracking analysis software.μg/mL to 2000 μg/mL were prepared by diluting albumin with PBS. The purified exosome samples lysed with RIPA lysis buffer were used as unknown samples. The standard and unknown samples were pipetted into the working reagent in the 96-well plate. Then, the 96-well plate was incubated at 37°C for 30 min, and the absorbance at 562 nm was measured on a microplate reader . The standard curve was constructed by plotting the absorbance for each albumin standard on y-axis against its concentration on the x-axis. Finally, the protein concentrations of the purified exosome samples were calculated according to the standard curve.The protein concentration of the isolated exosomes was determined with the BCA protein assay kit . Briefly, the working reagent was prepared by mixing reagent A and reagent B at a ratio of 50 : 1 and added to a 96-well plate. The standard samples with a work range from 20 μg exosomes diluted in the QIAzol lysis reagent. The expression levels of the eight selected miRNAs were detected by using miDETECT A Track™ miRNA qRT-PCR Starter Kit  with Bio-Rad CFX96 Real-Time System. The cycling parameters of the qPCR program were as follows: 95°C for 10 min (enzyme activation), followed by 45 cycles of 95°C for 2 s (denaturation), 60°C for 20 s , and 70°C for 10 s (extension). Primer sequences used for qPCR amplification of all the miRNAs were purchased from RiboBio. The miRNAs expression levels were normalized to cel-miR-39-3p and expressed as log10 (2−[CT (miR)–CT (cel-miR−39-3p)]). For all qPCR experiments, the samples were run in quadruplicate.Total RNA from the serum exosomes was isolated by using the miRNeasy Serum/Plasma kit  according to the manufacturers' protocol. To normalize RNA content, 1 pmol of exogenous cel-miR-39-3p  was added into per 100 http://mirwalk.umm.uni-heidelberg.de/). The filtering conditions of target genes are set by default. We also used this tool for functional annotation and signal pathway analysis of target genes.The prediction of miRNA target genes was performed by an online bioinformatics tool, miRwalk  curves were constructed for the differentially expressed miRNAs in the diagnosis of SCAD. P < 0.05 was considered to be statistically significant.Statistical analysis was performed using SPSS 24.0 statistical software , and the graphs were generated using GraphPad Prism 7.0. The normal variables were presented as mean ± SD and compared with the two-tailed Student Exosomes isolated from the serum of the control and SCAD groups were identified by TEM, western blot, and nanoparticle tracking analysis. TEM imagines showed that isolated exosomes had a bilayer membrane with a diameter of about 100 nm . NanoparThe clinical characteristics of the study population including 20 control subjects and 20 SCAD patients are shown in P = 0.09, and 0.28  vs. 0.29 , P = 0.16, respectively, The exosome protein concentrations of the control and SCAD groups were measured by the BCA method, and the total protein amount was calculated by multiplying the protein concentration by the volume of the isolated exosomes. There were no significant differences in the protein concentration and total protein amount between the exosomes from the control and SCAD groups  vs. 0.64 , n = 20) and SCAD group (n = 20) were analyzed by qRT-PCR. As shown in P = 0.038), miR-149-5p , and miR-32-5p  were significantly increased in the SCAD group compared to the control group  involved in cardiovascular diseases were significantly upregulated in the serum exosomes from SCAD patients, and these exosomal miRNAs had good predictive value for SCAD diagnosis. Furthermore, GO and pathway enrichment analysis revealed that some of the predictive targets of these miRNAs were involved in the pathophysiology processes of SCAD. Our findings suggest that exosomal miR-942-5p, miR-149-5p and miR-32-5p may sever as novel diagnostic biomarkers for SCAD.Increasing evidence suggests that the quantification of circulating exosomes could be used as a biomarker for some diseases , 31. AccFor several advantages, circulating exosomal miRNAs have emerged as promising biomarkers for various diseases. Firstly, miRNAs are stable and detectable in the circulating exosomes. Circulating miRNAs packaged in exosomes can escape from nuclease degradation due to the protection of the lipid bilayer membrane of exosomes and remain in a highly stable state . What isHowever, little was known about the potential of circulating exosomal miRNAs as biomarkers for CAD, especially SCAD. A previous study showed that the expression levels of exosomal miR-208a were increased in ACS patients and the high expression of miR-208a were associated with the decreased 1-year survival rate, suggesting that exosomal miR-208a could sever as biomarker for diagnosis and prognostic assessment of ACS . Moreoveα and ER stress marker expression [miRNAs have been recognized as important regulators in the development and progression of CAD , 40. Howpression . Moreovepression . As for pression . AnotherAtherosclerosis, the most common pathological substrate of CAD, is a chronic progressive process characterized by lipid deposition in the arterial wall. The pathogenesis of atherosclerosis is complex, involving endothelial dysfunction, macrophage adhesion and migration, VSMC migration and proliferation, oxidative stress, and inflammation . In the As far as we know, this study showed for the first time that circulating exosomal miRNAs had the potential as biomarkers for SCAD, which may provide an auxiliary tool for the diagnosis of SCAD and novel therapeutic targets for SCAD. Although some efforts were made in this study, several limitations should be taken into account. Firstly, in our study, three of the eight selected miRNAs were significantly upregulated in the circulating exosomes of SCAD patients, which suggested that our methods were effective. However, there might be other exosomal miRNAs with differential expression in SCAD patients, but these miRNAs were not included in our study. To identify as many differentially expressed exosomal miRNAs as possible in SCAD, it is advisable to explore the miRNA expression profile in exosomes from SCAD patients by using sequencing. Secondly, the serum exosomes were isolated and purified by using the ExoQuick reagent. Compared with ultracentrifugation, the ExoQuick reagent produced a higher yield but a lower purity of exosomes . HoweverIn general, our findings suggest that serum exosomal miRNAs such as miR-942-5p, miR-149-5p, and miR-32-5p may serve as novel diagnostic biomarkers for SCAD. Further studies are needed to reveal the function and mechanism of these exosomal miRNAs in the occurrence and development of SCAD.


Luffa is a kind of melon crop widely cultivated in temperate regions worldwide. Browning is one of the serious factors affecting the quality of Luffa. Therefore, the molecular mechanism of Luffa browning is of great significance to study. However, the molecular diversity of Luffa cultivars with different browning-resistant abilities has not been well elucidated. In our study, we used high-throughput sequencing to determine the transcriptome of two Luffa cylindrica cultivars ‘2D-2’ and ‘35D-7’. A total of 115,099 unigenes were clustered, of which 22,607 were differentially expression genes (DEGs). Of these DEGs, 65 encoding polyphenol oxidase, peroxidase, or ascorbate peroxidase were further analyzed. The quantitative real-time PCR (RT-qPCR) data indicated that the expression levels of the LcPPO gene (Accession No.: Cluster-21832.13892) was significantly higher in ‘35D-7’ compared with that in ‘2D-2’. Several POD genes  were also upregulated. Analysis of the plantTFDB database indicated that some transcription factors such as WRKY gene family may also participate in the regulation of Luffa browning. The results indicated that the divergence of genes expression related to enzymatic reaction may lead to the different browning resistances of Luffa. Our study will provide a theoretical basis for breeding of browning-resistant Luffa. Luffa (Luffa cylindrica), belongs to Luffa Mill. of Cucurbitaceae, is an annual herbaceous climbing plant  related to Luffa browning were identified. Expression levels of related genes were also validated and analyzed. Our results can provide potential insights into the molecular regulation of Luffa browning and a basis for cultivating improved Luffa germplasm with high browning resistance.The development of transcriptome sequencing techniques has led to identification of novel transcripts, splice isoforms, and expression differences in numerous species . A largeLuffa cultivars, ‘2D-2’ and ‘35D-7’, were preserved in the Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences and cultivated in the farm of Luhe Base . The field was approved for using by Jiangsu Academy of Agricultural Sciences (Contract No.: SC2018001). The fruits of each cultivar were collected after 10 days after pollination. The fruit pulps near the end of the stalk that are prone to browning were sampled for subsequent experiments. Samples were collected from three individual fruits. The samples were immersed in liquid nitrogen immediately and stored at −80 °C.The seeds of the two The total RNA of each sample was extracted according to the instructions of the plant RNA simple total RNA . Agarose gel electrophoresis and Agilent Bioanalyzer 2100  was used to detect the integrity of the RNA samples. RNA concentration and purity were measured by NanoPhotometer  and Qubit 2.0 Fluorometer .Construction of cDNA library was conducted in accordance with the instructions of NEBNext Ultra Directional RNA Library Prep Kit for Illumina . Ribosomal RNA was removed from total RNA to obtain the mRNA of the samples. Fragmentation buffer was added to break RNA into small fragments, which were used for the synthesis of the first-strand cDNA. Buffer, dNTPs , and DNA polymerase I were utilized to synthesize the second chain. The obtained double-stranded cDNA was purified by AMPure XP beads. After terminal repairing, A-tail adding, indexing adapter ligation, and fragment size selection, PCR amplification was conducted to enrich the short fragments for construction of the final cDNA library. The cDNA library was further quantified and its insert size was detected by Agilent 2100 to ensure that its quality could achieve the standard for sequencing. Sequencing was performed on the NovaSeq 6000 Sequencing System . Paired-end (150bp) sequencing was set as sequencing strategy.https://code.google.com/p/corset-project/) was used as unigene for subsequent analysis.Adapter-related reads, low-quality reads, and reads with relatively high number of base ‘N’ were filtered to ensure the accuracy of subsequent analysis. Clean reads after raw data filtering, error rate checking, and GC content distribution checking were used for subsequent transcript assembly. Trinity 2.6.6 was used to assemble the clean reads to obtain the reference sequence . The lonE-value ≤ 1E-5). The predicted amino acid sequences of the best aligning unigenes were transferred and searched by Pfam database using HMMER software to obtain the annotation information of the unigenes  , NCBI nounigenes . BLASTX P-value was conducted based on Benjamini–Hochberg method. The transcription abundance of the samples was expressed by fragments per kilobase of transcript per million fragments mapped (FPKM). The rationale of the DEGs was set as follows: —log2fold change—≥1 and the false discovery rate (FDR) < 0.05. The function of the DEGs was annotated by KEGG, GO, and COG database, and DEGs related to browning were identified according to the annotation information. Transcription factor annotation was identified by iTAK version 1.7 software in reference of plantTFDB . Assembly sequences has been deposited at TSA database under the Accession GILZ00000000. After data filtering, 9.67 G and 8.49 G clean high-quality reads were obtained. The percentage of Q20 and Q30 of ‘2D-2’ and ‘35D-7’ were 98.53% and 95.3%, 98.52% and 95.3%, respectively. The GC percentage in high-quality reads were 44.12% and 44.74%, respectively. The number of the contigs found by assembling the sequences according to Trinity was 144,048 with a mean length of 1,311 bp and an N50 length of 2,160 bp. After clustering, the longest clusters were identified as unigenes, and the total number of unigenes was 115,099 and the mean length and N50 length was 1,573 bp and 2,235 bp, respectively. The length distribution of the unigenes is shown in A total of 67,969,672 and 59,573,164 raw reads were detected by sequencing the two samples, ‘2D-2’ and ‘35D-7’, respectively. The sequencing raw data can be found in the Sequence Read Archive (SRA) database , and the detailed numbers of unigenes are listed in Luffa and related species  of COG database. Based on the orthologous and evolutionary relationships, homologous genes from different species were divided into different clusters and the function of unigenes was annotated . The topLuffa cultivars ‘2D-2’ and ‘35D-7’, a total of 22,607 DEGs were identified. Of the DEGs, 13,465 were down-regulated and 9,142 were up-regulated in ‘35D-7’. The overall distribution of the expression levels and annotation of DEGs was determined  was significantly higher in the Luffa cultivar ‘35D-7’ than in ‘2D-2’. Among the 45 DEGs related to LcPOD, 17 had lower expression and 28 had higher expression in ‘35D-7’ than in ‘2D-2’. In particular, the log2fold change of two POD genes  reached up to 12.16 and 11.10, respectively. Nineteen APX-related genes were also identified, but most of them were down-regulated in ‘35D-7’.To explore the gene regulatory mechanism of LcPPO, seven LcPOD genes, and four LcAPX genes were detected and analyzed to validate the expression levels of DEGs related to Luffa browning (LcPPO (Accession No: Cluster-21832.13892) in ‘35D-7’ was 57.09 times that in ‘2D-2’. Among the selected LcPOD genes, three  were upregulated significantly in ‘35D-7’. The expression levels of all four LcAPX genes were lower in Luffa cultivar ‘35D-7’ than in ‘2D-2’. In general, nine of the 12 genes exhibited consistent expression trends in the results of RT-qPCR and transcriptome analysis.The expression levels of some DEGs, including browning , their sLuffa is one of the most important melon vegetables, and its fruits are the main edible organ  were assembled in the fresh-cut Luffa cyindrica ‘Fusi-3’ fruits  Roem and analyzed their expression profiles. The PPO genes expressed in different tissues of Luffa and responded to the postharvest storage and fresh cutting conditions , POD, CAT  and SOD (encoding superoxide dismutase) may associate with the enzymatic browning of fresh-cut Luffa , peroxidase (POD) and ascorbate peroxidase (APX) were identified and further analyzed. The result of RT-qPCR confirmed the expression levels of 12 selected genes and demonstrated that these genes expressed higher in Luffa cultivar with strong browning resistance. Some transcription factors such as WRKY gene family may also participate in the regulation of Luffa browning. Our findings can establish foundation to cultivate improved Luffa germplasm with high browning resistance.Our study compared the transcriptomes of two 10.7717/peerj.9661/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9661/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9661/supp-3Supplemental Information 3Luffa unigene library, Figure S2: GO classification of the DEGs identified in the unigene libraries between two Luffa cultivars, Figure S3: KEGG classification of the DEGs identified in the unigene libraries between two Luffa cultivars. Table S3. Comparison of unigenes identified in transcriptome during browning of ‘Fusi-3’ fruits and transcriptomes of two Luffa cultivars ‘2D-2’ and ‘35D-7’. Table S4. Information of WRKY transcription factors identified in the transcriptomes of Luffa ‘2D-2’ and ‘35D-7’.Figure S1: GO classification of the Click here for additional data file.10.7717/peerj.9661/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9661/supp-5Supplemental Information 5Click here for additional data file.


The research was focused on developing a potentially antibacterial wound dressing made of polyurethane foam and loaded with bismuth-ciprofloxacin (Cip-Bi). The Cip-Bi chemical structure was confirmed by Fourier transform infrared spectroscopic (FTIR) analysis. The sought after antibacterial wound dressing was obtained by modification of the raw dressing with an iodine or bromine solution and subsequently with a Cip-Bi hydrogel. The amount of Cip-Bi loaded into the dressing matrix was determined indirectly on the basis of the differences in Cip-Bi concentrations, before and after the modification process, and the determination was performed with the HPLC (high-performance liquid chromatography) method. The modified dressing was found to have a two-step release of Cip-Bi, a feature helpful in the treatment of locally infected wounds and prevention of secondary bacterial infection. The zone of inhibition test against the selected Gram-positive and Gram-negative bacteria confirmed the antibacterial activity of the Cip-Bi-modified dressing. Preliminary tests conducted so far have been indicative of the Cip-Bi dressing’s relatively high activity against the tested organisms. Fluoroquinolones are representatives of an important group of chemotherapeutic compounds with a broad spectrum of activity against various pathogenic microorganisms resistant to aminoglycosides, penicillins, cephalosporins, tetracyclines and other antibiotics. In general, they exhibit activity against aerobic Gram-negative and Gram-positive bacteria, while they are especially active against the aminoglycoside-resistant Pseudomonas aeruginosa and beta-lactamase-producing organisms . Their mCombining metals with pharmaceutical agents can provide a strategy to improve drugs’ activity, decrease their toxicity and overcome resistance to their action. Quinolones, due to their ability to coordinate metal ions, may find a particularly interesting application in this strategy . TherefoCiprofloxacin (1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid) is a member of the second generation quinolone antibiotics family and it can be used in the treatment of complicated and uncomplicated urinary tract infections, lower respiratory tract infections, skin and skin-structure infections, urethral and cervical gonococcal infections, bone and joint infections, infectious diarrhea, typhoid fever and acute sinusitis . Due to E. coli (Escherichia coli) strain than the Gram-positive S. aureus (Staphylococcus aureus) strain.This paper presents the process of developing a new, wound healing, antibacterial dressing loaded with a ciprofloxacin-bismuth complex and its characterization, performed with the use of several analytical and microbiological methods. All this has resulted in producing a new type of wound dressing, composed of a hybrid material, which is capable of a gradual release of the ciprofloxacin-bismuth(III) complex. This newly obtained dressing exhibits an antibacterial activity both against the reference bacterial strains and clinical isolates from the infected wounds and appears to be more active on the Gram-negative Chitosan and its derivatives have been widely used in various fields of cosmetics and medicine including tissue engineering and wound dressings. Chitosan-based hydrogels could provide a moist wound environment, a protective role in secondary infections, remove wound exudate, and promote tissue regeneration and faster wound healing . ChitosaFor modification, foam material was used with a porous structure having the ability of solution absorption into open, narrow spaces by capillary action. Polyurethane foams are one of the most commonly used wound dressings. Polyurethane foam dressings belong to dressings that exhibit the ability to absorb exudates and provide a moist wound environment. By exudate management together with a better control of the pH and the oxygen permeability , these tBioactive dressing materials contain functional groups that can react with other molecules via hydrogen bonds and electrostatic interactions. As the studies have shown, the modification of the wound dressing leads to a decreased activity of proteases, such as metalloproteinases and elastase, in an attempt to stimulate wound healing .The wound healing, especially of chronic wounds, may be complicated by bacterial colonization. Generally, the infected wounds are treated with systemic antibiotics. Thus, the use of dressings containing antibacterial agents can be a valuable option to extinguish the infection in the course of wound healing. Ciprofloxacin, one of the fluoroquinolone group of antibiotics with a broad spectrum of activity, was used as the antibacterial protection for the dressing. Since metal-drug complexes are potentially capable of showing a greater antimicrobial activity when compared to the uncomplexed ligands, we decided to use the bismuth-ciprofloxacin complex instead of ciprofloxacin alone, surmising that it will raise the bioactivity against the resistant species . We alsoAs the literature has shown, quinolones represent the second most common antibiotic group associated with allergic reactions, which can be classified into 2 types: Ig-E-mediated immediate reactions and T-cell-mediated delayed reactions. Allergic sensitization to ciprofloxacin occurs relatively often (up to 30% of anaphylaxis and 35% of delayed hypersensitivity) . So, theIt is likely that in a wound, hydrolysis of the complex with the release of bismuth may occur. It is also possible that a compound containing bismuth will treat the wound as well as inhibit inflammatory reactions. Therefore, preliminary skin monitoring tests were carried out following application of the modified samples of the dressings. This was conducted with the use of the skin analysis system with photographic documentation. The results confirming the therapeutic potential of the modified dressings will be shown in the later work.−1 (O-H stretching vibrations of carboxyl group), at 3100–3012 cm−1 (C-H stretching vibrations of the aromatic ring), at about 2929 cm−1 , at 2691–2461 cm−1 (the region of stretching vibrations of the ionized amine groups) and the intense absorption bands at 1701 cm−1 and 1624 cm−1 related to the stretching vibrations of carboxy carbonyl (C=O of -COOH) and carbonyl (C=O) in position 4 of the quinoline ring respectively, as well as the absorption bands in the region of 1510–1446 cm−1, which correspond to the C=C stretching vibrations of the aromatic ring. The Cip-Bi spectrum compared to the Cip spectrum shows the absence of a peak at 3528 cm−1 and a peak at 1701 cm−1, and the appearance of peaks at about 1575 and 1479 cm−1, due to the symmetrical and asymmetrical carboxylate stretching vibrations (COO-), as well as the peak of out-of-plane bending vibrations (COO-) of medium intensity at about 830 cm−1, while the peak of the carbonyl group (4-oxo) has slightly shifted toward the lower frequencies (from 1624 to 1613 cm−1). The observed changes provide evidence for the substitution of protons in the carboxylic group of ciprofloxacin (deprotonation) by a Bi-containing cation. Bismuth is chelated to the ciprofloxacin molecule via the Bi-O bond through the carboxylate anion and the O → Bi bond through the 4-oxo group. The obtained spectra are similar to the spectrum reported by Shaikh et al. [The spectrum of Cip  is compah et al. .The complexation process by influencing the electron transfer in the chromophore caused a modification of the UV (ultraviolet) absorption spectrum of the Cip molecule. The spectral effects associated with a wavelength shifting, peak disappearance and changes in the intensity of the bands characteristic of Cip were observed. The absorption spectrum of Cip in an acidic environment shows a strong absorption band at 277 nm and two much less intense bands at 312 and 328 nm B. Upon cThe increase in absorbance was proportional to the increase in molar concentration for both Cip and Cip-Bi . Molar absorptivity was determined to evaluate the number of moles attached to the bismuth atom in the Cip-Bi complex . The moln = 18) was constructed by plotting the peak areas of Cip-Bi standard solutions versus the concentrations in the range of 3.0–18.0 μg/mL with the correlation coefficient (R) greater than 0.9995. A typical linear regression equation for the calibration curve was y = 69,898 ± 412x − 45,270 ± 4808 (R2 = 0.9994) for HPLC with UV-detection, or y = 42,926,873 ± 234,546x − 14,396,984 ± 2,740,277 (R2 = 0.9995) for HPLC with FL-detection. The HPLC method was developed  for the The detection and quantification limits (determined on the basis of the standard deviation of residues of the linear regression curve obtained for a dilution series of 3.0 μg/mL standard solution) were found to be 0.04 and 0.11 μg/mL for FL-HPLC or 0.12 and 0.37 μg/mL for UV-HPLC, respectively. The intraday and interday precision estimated by successive injections of three individual standard samples containing 5, 10 and 15 μg/mL of Cip-Bi were varied in the range of 0.39–2.77% and 2.52–4.46% , respectively.The HPLC method  was satiIn order to prevent the secondary bacterial infection, an ideal wound dressing should be expected to release an antibacterial agent. Controlled drug release provides localized delivery and minimizes the risk of systemic side effects. Polymer-based dressings such as polyurethane foams and hydrogels are often used for controlled drug delivery to wounds. Drug release from polymeric carriers is mostly caused by hydration of the polymer, swelling, drug diffusion through the matrix or matrix erosion. In practice, most of the wound dressings release the drugs embedded in the matrix by a combined mechanism . The proIt was found that the majority of bacteria that cause wound infections are resistant to a minimum of one of the most commonly used antibiotics. Moreover, infectious strains become resistant to almost all classes of antibiotic, which makes it necessary to look for new substitutes . Since tE. coli) and Gram-positive (S. aureus) bacteria. First, the MICs (minimum inhibitory concentrations) values of the synthesized Cip-Bi complex as well as the parent components, i.e., Bi (bismuth citrate) and Cip (ciprofloxacin hydrochloride), were evaluated. As E. coli than S. aureus. Significant differences in antibacterial activity of these compounds against E. coli and S. aureus strains were confirmed by Tukey’s test: p (0.002–0.03) < 0.05. In our study, the samples were tested for activity against selected medically relevant pathogens, including, what is important, clinical strains: Gram-negative  against h higher . E. coli bacteria .The complexation with bismuth resulted in an intensification of the activity of ciprofloxacin against The antibacterial activity of Cip-Bi-loaded dressing samples was subsequently evaluated using the plate antimicrobial test in the solid medium. The obtained results are presented in S. aureus and about five times more active than the iodine-treated dressing against E. coli. The zones of inhibition, on the Mueller Hinton agar, around the modified dressings containing approximately 0.4 mg of Cip-Bi, were 19.5–36.0 mm. The untreated dressing (sample (S)1), similarly to the dressing activated with bromine (S2) or iodine (S3), showed no inhibition zones against the reference strains. The bromine-activated dressing loaded with hydrogel (S4) showed an insignificant activity, whereas the activity of the dressing with hydrogel but activated with iodine (S5) was slightly higher against both bacterial species . However, the antibacterial activity of the Cip-Bi-loaded dressings  was by far the largest, as it is illustrated in p (0.0002–0.02) < 0.05). A significant increase in antibacterial activity for the Cip-Bi hydrogel-loaded dressings  in relation to the hydrogel-loaded dressing without Cip-Bi complex (control samples: S4 and S5) was confirmed by Tukey’s test  showed the highest inhibitory activity against all the tested clinical strains, while the bromine-activated, Cip-Bi-modified dressings’ activity was slightly lower (S6). It is worth noting that both tested hybrid biomaterials  were effective against the reference as well as clinical strains. However, in the case of clinical strains, similarly to the reference strains, the tested Gram-negative bacteria were less resistant to the activity of the modified dressings than Gram-positive bacteria. The present study confirms an increased antibacterial activity of the Cip-Bi-loaded dressings in comparison to unmodified dressings. Apparently, the CIP-Bi-loaded dressings exhibit a high antimicrobial potential and may effectively protect wounds from infections. Pure ciprofloxacin (Cip) as hydrochloride was obtained from Polfarma . Chitosan  and poly(ethylene glycol), PEG-200, were purchased from Sigma-Aldrich Co. . Biatain® non-adhesive polyurethane foam dressing  was supplied by a local medical supplier. Bismuth(III) citrate (Bi), iodine, bromine, triethylamine, hydrochloric acid (0.1 M), fixanal 1 L Ampoule (from Sigma-Aldrich Co.) and 25% ammonium hydroxide solution  were also purchased. Methanol and acetonitrile (both of HPLC grade), 35–38% hydrochloric acid, 85% orthophosphoric acid and other reagents and solvents of analytical grade were acquired from Avantor Performance Materials, Gliwice, Poland S.A. The water used in the experiments was double-distilled.v/v) and mixed by constant stirring for about 5 h at 50 °C. The precipitated complex was filtered out and washed with a mixture of methanol-water (1:1 v/v) until the filtrate was free of Cip in TLC (thin layer chromatography). The obtained product was dried under vacuum.The bismuth-ciprofloxacin complex was synthesized as a result of the bismuth reaction with ciprofloxacin performed in compliance with the previously described procedure  and the −1 at the spectral resolution of 4 cm−1. All spectra were averaged over 32 scans.The chemical structure of Cip-Bi was examined with attenuated total reflection–Fourier transform infrared spectroscopy (ATR–FTIR) using a Thermo Scientific Nicolet 6700A spectrometer  equipped with a single-bounce beam path, a diamond crystal at a 45° angle of reflection and OMNIC 8.1 computer software. The Cip-Bi with respect to Cip was analyzed over the region from 600 to 4000 cmThe absorption spectral measurements were carried out on a UV-vis Hitachi U-2001 spectrophotometer  controlled by UV Solutions software using 1 cm matched quartz cells. The solutions of Cip and Cip-Bi in the concentration range of 5–20 μg/mL  were prepared in 5% hydrochloric acid. The ultraviolet spectra were recorded in the range 200–400 nm. An accurately weighed amount of 0.2 g of Cip-Bi compound was dissolved in 10 mL of 10% nitric acid and heated in a boiling water bath for 5 min. Next, 0.1 g of potassium chlorate was added, the solution was heated for another 5 min and 100 mL of a distillate water was added. The solution was titrated with 0.05 M sodium edetate against xylenol orange as an indicator.The samples of the polyurethane foam dressing of about 0.5 × 0.5 cm in size were modified using a two-step approach. Initially, the samples were activated using two activating agents and next, they were loaded with a hydrogel containing ciprofloxacin-bismuth complex (Cip-Bi hydrogel). In order to obtain the Cip-Bi hydrogel, 5 mg of chitosan and 15 mg of bismuth-ciprofloxacin complex were dissolved in 10 mL of 10% citric acid, then 5% glutaraldehyde and PEG-200, each in the amount of 0.3 mL, were added and a mixture was made up to 15 mL with ethanol (Cip-Bi concentration was 1 mg/mL). In the method 1, the samples were gently stirred with a 1% ethanolic iodine solution for 1 h at 37 °C. The excess solvent was removed by placing the samples on the filter paper. Next, the samples were treated with the Cip-Bi hydrogel. In the method 2, the samples were prepared in the same manner, but using a 1% ethanolic bromine solution as an activating agent. The samples subjected to further qualitative tests were previously dried at 37 °C.v/v/v/v) as the mobile phase at a flow rate of 0.7 mL/min.Chromatographic analysis was carried out at λ = 279 nm (spectrophotometric (UV) detection) and at λex 279 nm/λem 430 nm (fluorescence (FL) detection) on the Waters, e2695 Aliance, HPLC system  equipped with the Purospher STAR RP-18, 4 × 125 mm (5 µm), column , and the Waters Empower Pro v.2 software for acquiring, processing, reporting and managing the chromatographic information using a mixture of triethylamine–acetonitrile–methanol–phosphoric acid (10 mM)  were used for the determination of the Cip-Bi analyte, and calibration curves were generated on the basis of the obtained data. This test was conducted for the foam dressing samples  in order to check how much Cip-Bi was loaded into the samples. The dressing samples activated with iodine or bromine solution were added to the Cip-Bi hydrogel solution (containing 1 mg of Cip-Bi per 1 mL) and gently stirred at 37 °C. The samples of Cip-Bi hydrogel solution, before and after modification of the dressing, were diluted in the 1:100 ratio, filtered through a 0.45 membrane filter and subsequently determined with the use of the UV-FL HPLC method. The concentration of Cip-Bi in the hydrogel solutions was calculated from the calibration equation after multiplying by the dilution factor. The quantity of Cip-Bi hydrogel solution absorbed by the dressing sample was evaluated from the volumes of the Cip-Bi hydrogel solution, before and after dressing modification.The Cip-Bi release study conducted in vitro was to estimate the releasing profile of Cip-Bi from the drug-loaded samples . The Cip-Bi-loaded samples were immersed in 2 mL of 0.1 mol/L hydrochloric acid and shaken repeatedly every 30 min with a fresh portion of the washing solution at 37 °C. The quantity of Cip-Bi in the washing solutions was determined directly by the UV-FL HPLC method and calculated from the calibration equation.Escherichia coli ATCC 25992) and Gram-positive bacterium (Staphylococcus aureus ATCC 25923). The clinical bacterial isolated  from infected wounds or urine were obtained from the University Hospital patients and stored in the strain bank at the Department of Biochemistry and Biotechnology, the Medical University of Lublin, Poland. The stock cultures were maintained at −70 °C in the Tripticase Soy broth with 16% (v/v) glycerol until the study was performed. Before the experiments, each bacterial strain was passaged onto a fresh Mueller-Hinton agar (M-H)  at 30 °C for 48 h. Next, the inocula were prepared with fresh microbial cultures in sterile 0.9% NaCl to match the turbidity of 0.5 McFarland standard. The antibacterial activity of the samples against the tested bacteria was evaluated by measuring the zones of inhibition in the disk diffusion method (Kirby-Bauer Disk Diffusion Susceptibility Test Protocol). Antibacterial disc diffusion assays were carried out on Petri plates with solid medium (M-H agar). Appropriate strain cultures were separately spread over the agar surface using a cotton swab. Next, the tested liquid samples, i.e., Bi in 10% hydrochloric acid (10 μL of 2.0 mg/mL solution), Cip and Cip-Bi in 10% citric acid (10 μL of 2.0 mg/mL solution), or previously prepared pieces of the modified dressing with about 0.4 mg of active substance, were placed on a sterile disc or directly on the Petri plates containing the agar medium. After 18 h of incubation at 37 °C, the zones of microbial growth produced around the tested samples were measured and recorded as the diameters of inhibition (mm). The unmodified material polyurethane foam dressing (Coloplast) was tested as a negative control (S1), while the dressing activated with a bromine solution (S2) or iodine solution (S3), both loaded with the hydrogel without Cip-Bi complex , were used as positive controls in the microbial tests.The zones of bacterial growth inhibition caused by the tested samples, previously modified in compliance with the methods 1 and 2 , were evaluated for the reference microorganisms from the American Type Culture Collection (ATCC), including Gram-negative bacterium (6 CFU (colony-forming unit)/mL. After incubation (37 °C for 18 h), the panel was digitally analyzed at 600 nm using the hybrid multi-mode microplate reader  with the dedicated software system. The growth intensity in each well was compared with the controls: negative (sterile broth), positive  and dye control (broth with tested sample without bacteria).In order to obtain final concentrations of the tested samples, ranging from 0.00061 to 40 µg/mL (in case of Cip and Cip-Bi) or from 0.00611 to 400 µg/mL for Bi, the Minimal Inhibitory Concentration (MIC) value was determined , 2018) with the M-H bullion , using their serial two-fold dilutions. As the control background, the antibacterial activity of the used solvents was evaluated (10% HCl for Bi and 10% citric acid for Cip and Cip-Bi). The sterile 96-well polystyrene microtitrate plates were prepared by dispersing 200 µL of appropriate dilution of the sample in a culture broth per well. Finally, 2 µL of a bacterial inoculum were added to the wells to obtain a density of 1.5 × 10p < 0.05 level.The results are expressed as mean ± RSD (relative standard deviation). Microbiological testing results were evaluated statistically by analysis of variance  and statistical significance was set accordingly at the The threat of an increase in the number of bacteria resistant to classic antibiotics, including Cip, made us look for other solutions to inhibit the growth and multiplication of pathogens. It seems that such an alternative to a conventional Cip could be the Cip-Bi complex. Moreover, the combination of Cip with Bi may potentially reduce ciprofloxacin allergic reaction by reducing its absorption. The findings presented in this paper allow to conclude that the Cip-Bi-modified hydrogel dressing, obtained in an uncomplicated way, is a new wound dressing with potential wound healing properties in comparison to the unmodified dressings. We were able to incorporate the Cip-Bi complex into the dressing matrix successfully, which is confirmed by the obtained antimicrobial test results. The newly developed dressings show activity against tested reference and clinical Gram-positive and Gram-negative strains. Yet, in order to evaluate their potential application in practice, further research into this issue should focus on their safety and efficacy. It should be noted that there is insufficient evidence as yet to confirm the effect of antimicrobial dressings on wound healing in clinical practice.


Inconsistent use of generic and disease-specific health-related quality of life (HRQOL) instruments in multiple sclerosis (MS) studies limits cross-country comparability. The objectives: 1) investigate real-world HRQOL of MS patients using both generic and disease-specific HRQOL instruments in the Netherlands, France, the United Kingdom, Spain, Germany and Italy; 2) compare HRQOL among these countries; 3) determine factors associated with HRQOL.A cross-sectional, observational online web-based survey amongst MS patients was conducted in June–October 2019. Patient demographics, clinical characteristics, and two HRQOL instruments: the generic EuroQOL (EQ-5D-5L) and disease-related Multiple Sclerosis Quality of Life (MSQOL)-54, an extension of the generic Short Form-36 (SF-36) was collected. Health utility scores were calculated using country-specific value sets. Mean differences in HRQOL were analysed and predictors of HRQOL were explored in regression analyses.n = 88; France: n = 58; the United Kingdom: n = 15; Spain: n = 10; living elsewhere: n = 11). Mean MSQOL-54 physical and mental composite scores  were lower, whereas the SF-36 physical and mental composite scores  were higher than reported in previous clinical trials. The mean EQ-5D utility was 0.65 (SD:0.26). Cross-country differences in HRQOL were found. A common predictor of HRQOL was disability status and primary progressive MS.In total 182 patients were included (the Netherlands: The effects of MS on HRQOL in real-world patients may be underestimated. Combined use of generic and disease-specific HRQOL instruments enhance the understanding of the health needs of MS patients. Consequent use of the same instruments in clinical trials and observational studies improves cross-country comparability of HRQOL.The online version contains supplementary material available at 10.1186/s13690-021-00561-z. Multiple sclerosis (MS) is a chronic disease with neurological dysfunction of the central nervous system, affecting 700,000 people in Europe and some 2.3 million people worldwide , 2. WhilMany different generic or disease-specific HRQOL instruments have been used in MS-related randomized controlled trials (RCTs), observational studies and patient registries , 7. WhilThe use of diverse HRQOL instruments in different MS studies limits the comparability of outcomes between these studies . To the We performed a cross-sectional online survey between June and October 2019 in six European countries . These countries were selected to give a representative overview of the HRQOL of the EU-5 and the Dutch MS patient population. Patients were recruited through the information channels of national patient societies and social media. Based on feasibility and the exploratory nature of the study  we aimed to include 50 persons per country (i.e. non-probability sampling). In France the patients were also recruited by a MS specialist working at a MS centre in Paris. The study information and informed consent form could be downloaded by the patient. The research protocol was reviewed and approved by the Medical Research Ethics Committees of the Erasmus Medical Centre (MEC-2018-1636). Qualtrics XM software was used to perform the survey. The STROBE checklist for cross-sectional studies was used for reporting this study .Patients had to be ≥18 years old and have the diagnosis of clinically definite MS , relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), or primary progressive MS (PPMS)). No restrictions were made on whether patients were currently taking or had previously taken disease modifying therapies (DMTs). Participants required access to the internet.The online web-based survey consisted of three parts: 1) patient demographics and clinical characteristics; 2) the EQ-5D with five levels (EQ-5D-5L) to collect information about the patient’s health state ; and 3) The survey was available in six languages and the participant was free to choose which language to use when completing it. Questions regarding demographics and clinical characteristics were translated from Dutch into the other languages by a professional translation agency and double-checked by native speakers. Official translations of the EQ-5D-5L and the MSQOL-54 were used.Patients were asked to provide information on their age, country of residence , nationality, gender, marital status and educational level. Clinical characteristics included the type of MS, age at diagnosis, disability and their current and previous treatment. Disability was self-reported using the Expanded Disability Status Scale (EDSS), an instrument to rate neurological impairments, with a total score ranging from 0  to 10 (death due to MS) .The EQ-5D-5L is a standardized and validated HRQOL instrument , 16 thatThe MSQOL-54 is a validated instrument with an adequate test-retest reliability, construct validity and internal consistency . The insp < 0.05.Statistical analyses were performed with Stata 16.0. Analyses were stratified according to country of residence. Differences in mean scores across countries were calculated using the analysis of variance (ANOVA) test; if assumptions for the ANOVA were violated (we checked the distribution of the variables by plotting a histogram), the non-parametric Kruskal-Wallis test was used. Testing for a relationship between categorical variables was done using the chi-square test or the non-parametric Fishers exact test for small samples. Post-hoc analysis was done using either the Bonferroni correction or the Dunn’s test. Statistical significance was defined as Bivariate association of the EQ-5D-5L dimensions to EDSS was investigated with Spearman correlation coefficients. Univariate and multivariate analyses were conducted to examine the relationship between patient demographics and the EQ-5D-5L dimensions, health utility, the MSQOL-54 scales and the PHCS/MHCS. Covariates included in the multiple regressions were based on significance in the univariate analysis. A linear or logistic regression was performed for a continuous or categorical outcome variable, respectively. The stepwise backward selection method was used for the multivariate regression and variables were kept in the model based on significance.n = 55), did not give informed consent (n = 39), had provided an age of diagnosis that was older than their current age (n = 3), or were younger than 18 years (n = 2). This left 182 participants for analysis.The attempted sample size of 50 per country was not feasible in the UK, Spain, Germany and Italy given that patients were very difficult to recruit, therefore we ceased recruitment 5 months after the start of the study. A total of 281 patients were recruited and started the survey. Ninety-nine participants were dropped from the analysis since they did not finish the survey . The total population had a median age of 43 years old and the average age at diagnosis was 34 years old. Roughly 80% were female and most participants (80%) had RRMS. There were no significant differences in patient demographics across the countries other than the age at diagnosis (range: 31.2–41.6 years) and educational level (an average of 52% having a university degree).Patient demographics can be found in Table n = 131, 72.0%) or had received it in the past . Of those currently taking a DMT, 44.7% were taking a first-line DMT, which was either an injectable  or an oral treatment . Over half of the patients currently taking a DMT were receiving a second-line therapy . A minority received oral treatment  and the majority received infusion therapy . Many of the patients receiving infusion therapy were taking ocrelizumab .The results regarding current and past treatments can be found in the Additional file p = 0.018) and ocrelizumab (p = 0.003)  and 44% had moderate problems with usual activities. A majority had slight to moderate pain and discomfort . Generally, the patients had no problems with self-care  or anxiety and depression .The total mean health utility score was 0.65 SD:0.26) (Table 6 (Table p < 0.001) (Table p = 0.012) (results not shown). No statistical between-country differences were found amongst the EQ-5D-5L dimensions  Table . Howevern = 28; 62.2%) or self-care  . Patients with greater disability (EDSS 3–5.5 and ≥ 6) were more likely to have moderate to severe problems in mobility, daily activities and pain/discomfort. However, disability was not associated with anxiety/depression. Furthermore, there was a strong and significant correlation of the EQ-5D-5L domains mobility, self-care and usual activities to disability  generally had no problems with mobility (3%) Fig. . HoweverFig. n = 9; 42.2%.p = 0.017). In contrast, no significant difference between countries was found on the MSQOL-54 MHCS . The mean SF-36 physical composite score (SF-36 PCS) and mental composite score (SF-36 MCS) for the total population was 46.8 (SD: 22.6) and 53.1 (SD: 22.45), respectively. Both scores differed significantly between countries .The results of the MSQOL-54 and SF-36 are presented in Table Table Additional univariate and multivariate models for the EQ-5D-5L dimensions and utility, MSQOL-54 scales and composite scores are shown in the Additional file The aim of this study was to examine real-world HRQOL of patients with MS in several European countries. The generic health utility instrument (the EQ-5D-5L) and the hybrid disease-specific MSQOL-54 (including the SF-36) instruments were used to calculate HRQOL. Compared to previous research, our results indicate that the HRQOL of MS patients may have been overestimated. We found a relatively low health utility score, with no between-country differences amongst the EQ-5D-5L dimensions. Somewhat low HRQOL was found using the MSQOL-54 with between-country differences. Furthermore, disability status and PPMS is negatively correlated with HRQOL.The mean EQ-5D utility score 0.65 ± 0.26 SD) in our population was lower than the scores (range: 0.69–0.78) reported in other multi-country studies –26. Prev SD in ouOur European population had a somewhat higher mean SF-36 physical component summary score (46.8 SE:1.7) and mental component summary score (53.11 SE:1.6) than most previously published DMT studies. The CONFIRM and DEFINE study, examining HRQOL while taking dimethyl fumarate, found mean SF-36 PCS and MCS of 43.1 and 47.2, and 43.4 and 45.3, respectively , 25. AlsThe mean MSQOL-54 composite scores  in our European population were 10–20 points lower than scores reported in previous observational studies –32. For Disease-specific measurements are more extensive and have greater sensitivity to changes, meaning it can detect HRQOL differences between patients . When coWe want to inform physicians and policy makers that it is useful to include HRQOL instruments in clinical practice and in clinical trials. The use of such instruments in clinical practice enables physicians to know what dimensions of HRQOL to target. As such, it is possible to set up a personalized treatment plan, together with the patient, based on the HRQOL results . MoreoveThere are various limitations to our study worth noting The design of this study was to examine HRQOL in Europe, and not between-country differences. However, post-hoc analysis revealed some between-country differences in HRQOL. Possible explanations of between country differences in HRQOL may be explained differing healthcare systems, the recruitment method and small samples in the UK and Spain. The healthcare systems of the examined countries differ, thereby possibly affecting the quality of care received by patients. Patients were recruited via the information channels of national patient societies, social media and via an MS specialist in France. Therefore, we had no control over how and to whom the patient societies reached out too, or the reach of our social media channels within the MS community. It is possible that the patient societies differed in how actively they promoted the study, leading to selection bias. Furthermore, the small samples from the United Kingdom and Spain limits their representability of the HRQOL status of those two countries, thus caution is needed when making statements on their HRQOL. Since we had small samples, we did not perform regressions per country. If larger samples had been recruited this may have given insight into possible country differences. Future studies should be designed to specifically examine cross-country differences and controlling for more information than we have included in this study.Many disease-specific HRQOL instruments have been developed since the introduction of the MSQOL-54 in 1995, such as the Functional Assessment of MS (FAMS), the Hamburg Quality of Life Questionnaire in MS (HAQUAMS), the MS Impact Scale-29 (MSIS-29) and the MS International Quality of Life questionnaire (MusiQoL) to name a few , each foOne MS centre in France was involved in the data collection and this may have led to sampling bias. This reduces the comparability of the French patients to patients in the other countries, although it is comparable to a previous French multicentre study by Lebrun-Frenay et al. (2017). For example, both studies found that patients were in their early thirties when diagnosed, at least 50% of the patients had a university degree and the type of DMT used was similar . The simThree patients (other than patients living elsewhere) filled out the survey in a language that differed from the official language of their country of residence which may have impacted health utility results  , 43. AllSince the EDSS was self-reported, it is possible that some patients incorrectly estimated their EDSS due to a lack of understanding of the scale, despite having the option to indicate that they did not know it. However, the negative association we observed between EDSS and HRQOL suggests that this issue had a limited effect on the results. Nevertheless, caution is needed when interpreting the results. At the time of data collection the self-reported disability status scale (SRDSS) as a proxy measure to estimate EDSS was not yet available, however such a measure may be useful for self-assessment of disability in an online questionnaire environment . AnotherOur results indicate that, till now, the effects of MS on HRQOL may have been underestimated in real-world MS patients. The combined use of both generic and disease-specific HRQOL instruments as outcome measures in clinical trials and observational studies allow for a deeper understanding about specific health needs of MS patients. To enhance the comparability of cross-country data from RCTs, observational studies, or patient registries it is essential to use the same instruments consequently. This study has made a first attempt to do so across Europe, however a more collective effort has to be made by all persons involved in health care research.Additional file 1.


Opioid dependence carries the highest disease burden of all illicit drugs. Opioid agonist therapy (OAT) is an evidence-based medical intervention that reduces morbidity and mortality. There is limited knowledge on the health-related quality of life (HRQoL) of long-term patients in OAT. This study measures HRQoL and self-perceived health of long-term patients on OAT, compares the scores to a Norwegian reference population, and assesses changes in these scores at 1-year follow up.We conducted a nested prospective cohort study among nine OAT outpatient clinics in Norway. 609 OAT patients were included, 245 (40%) followed-up one year later. Data on patient characteristics, HRQoL, and self-perceived health was collected. HRQoL was assessed with the EQ-5D-5L, which measures five dimensions  on a five-point Likert scale (from “no problems” to “extreme problems”). An UK value set was applied to calculate index values (from 0 to 1) for the EQ-5D-5L and compare them to a Norwegian reference population. Self-perceived health was measured with EQ-VAS (from 0 to 100).Mean (standard deviation (SD)) EQ-5D-5L index value at baseline was 0.699 (0.250) and EQ-VAS 57 (22) compared to 0.848 (0.200) and 80(19) for the Norwegian reference population. There were large variations in EQ-5D-5L index values, where 43% had > 0.8 and 5% had < 0.2 at baseline. The lowest EQ-5D-5L index values were observed for female patients, age groups older than 40 years and for methadone users. At follow-up, improvements in HRQoL were observed across almost all dimensions and found significant for mobility and pain/discomfort. Mean (SD) overall index value and EQ-VAS at follow up were 0.729 (0.237) and 59 (22) respectively.The average HRQoL and self-perceived health of OAT patients is significantly lower than that of the general population, and lower than what has been found among other severe somatic and psychiatric conditions. Around 34% had very good HRQoL, higher than average Norwegian values, and around 5% had extremely poor HRQoL. Opioid dependence is a severe chronic relapsing disorder consisting of a cluster of physiological, behavioral, and cognitive phenomena . WorldwiIncreased focus on, and availability of harm reduction programs, such as OAT have lowered the demand for heroin in Western Europe including Norway . OAT is Factors associated with poor HRQoL among OAT patients are older age, female gender, and mental and physical comorbidity , 26, 27.The principal aim of this study is to evaluate the HRQoL and self-perceived health of a large cohort of long-term patients with opioid dependence enrolled in an integrated OAT program in Norway. The HRQoL of OAT patients will also be compared to that of the general population in Norway. Finally, an assessment of changes in HRQoL and self-perceived health at one-year follow-up will be conducted.This study is a nested prospective cohort study linked to the Integrated Treatment of Hepatitis C study (INTRO-HCV) . The obsThe study sample included individuals diagnosed with opioid dependence according to International Classification of Diseases version 10 (ICD-10) , currentThe EQ-5D-5L instrument is a widely used generic measure of HRQoL  and valip values as the dependent variable. More weight was given to cases with valid data, which were associated with highest probability of having missing data, and less weight was given to cases with lowest probability of missing. The mean for the population weights was 1.0 (SD 0.12) in our model. Statistical significance was set at p < 0.05 level. All analyses were made with STATA SE 16.0.Responses to the five HRQoL dimensions are coded as a five-digit code, which represents a numerical description of a health state. The digits have no arithmetic properties and therefore a single summery number  needs to be arrived by applying a formula with an appropriate value set, which is a representative sample of the general population. The index value then represents how good or bad a health state is according to the preferences of the general population, ranging from 1  to 0  . In the The patients were predominantly male 71%) with a mean (SD) age of 44 years (10). The age range of the study sample was 23–74 years. Most received buprenorphine-based medication (60%) followed by methadone (38%). Duration in OAT treatment ranged from 0 to 25 years, with a mean (SD) of 7.9 (5.4) years. Results therefore reflects HRQoL of patients that have received OAT over a long period of time. Of the participants, 45% had completed secondary school and 40% completed high school. Almost 80% received either disability pension or social benefits as main source of income. Of the 152 OAT patients with children under 18 years, 21% reported they had no visiting rights  EQ-5D-5L index value for OAT patients was 0.699 0.250) at baseline. Forty-three percent had an index value above 0.8, meaning they had “no problems” in the five health domains. Thirty-four percent of the sample even had an index value above 0.848 (Norwegian reference population [40]), meaning their HRQoL was better than that of the Norwegian general population. Around 5 % had an index value below 0.2, meaning they had “extreme problems” in HRQoL. The distribution in baseline EQ-5D-5L index values are shown in the Pen’s Parade  EQ-VAS score of OAT patients was 57 (22) for the total sample at baseline, meaning their self-perceived health was considerably lower compared to the Norwegian reference population of 80 (19) . Femalesn = 245) are indicated with vertical lines. For instance, a patient with an index value of 0.563 at baseline and a long vertical line going up to 0.840 at follow-up means this patient reported a significant improvement in HRQoL. A patient with a vertical line going down from baseline shows worsen HRQoL between baseline and follow-up. Patients with no follow-up data (n = 364) or no change at follow-up (n = 26) has no vertical line. Figure Altogether 245 (40%) of the 609 patients at baseline were included for the follow-up analyses. As shown in the Pen’s Parade  and for both genders , age group 26–40 (p = 0.002) and buprenorphine-based patients (p = 0.027) as shown in Table p = 0.008) and pain/discomfort (p = 0.025) .Significant improvement in self-perceived health (EQ-VAS) were found for males  were found in many of the OAT patients. However, large variations in EQ-5D-5L index values were found between individuals, both at baseline and at follow-up. Significant improvement in overall HRQoL was observed at one-year follow-up with around half of the OAT patients reported some improvement in HRQoL while around one-third experienced worse HRQoL at one-year follow up, with great individual variations. Males reported significant improvement in their self-perceived health.Compared to the general Norwegian population, which reported no problems regarding mobility (85%), self-care (98%), usual activities (82%), pain/discomfort (54%) and anxiety/depression (79%) , OAT patet. al (2019) study of a large OAT cohort in Germany; found that OAT patients had a lower HRQoL than the general population [Our findings are consistent with prior research, such as Strada pulation . Severalpulation , 27. Howpulation . In our pulation , 42. Incpulation . It is aPrevious research has revealed that HRQoL improves considerably at OAT treatment initiation and the first few months : howeverHRQoL in the long-term OAT population was lower in average compared to what is found in other patient groups, such as diabetes type 1 and 2 , HIV/AIDFurthermore, as HRQoL profiles of OAT patients are diverse and dynamic this has implications for personalized patient care and the need for regular assessment of HRQoL as an outcome. We need to better understand what drives the extreme and rapid changes in HRQoL in both positive and negative directions among OAT patients. We need to know how to best prevent large drops in index score and how to increase and maintain the increases in index score over time. Additionally, females have worse HRQoL scores compared to men, which indicates that OAT programs should particularly focus on how to improve HRQoL of females and find explanations for why females have lower HRQoL. Similarly, we found that patients older than 40 years have worse HRQoL. This shows that we need to re-examine health care needs of older patients are met and how we can address their needs better. This is particularly important as long-term OAT patients are now aging and we need to plan for aging populations receiving OAT.A strength of this study is the large sample size of long-term OAT patients at baseline who are receiving the same level of integrated OAT treatment across their respective OAT outpatient clinics in the two cities. However, there are also limitations to our study. Follow-up was conducted on a sub-group of the initial sample. To reduce the selection bias due to the loss-to-follow-up we performed an inverse probability weighted method. In general, it is problematic to compare HRQoL between studies as setting, population, and level of OAT integrated treatment varies and different instruments are being used. This is also the case for comparisons between results based on EQ-5D-3L and EQ-5D-5L, and when different national value sets are being used. Comparative performance across patient groups is driven by differences in the descriptive systems and associated value sets  Another We found considerably lower HRQoL among long-term OAT patients in average compared to the general Norwegian reference population. However, this is a heterogeneous population. Around one-third had very good HRQoL, higher than average Norwegian values. Improvements in HRQoL were found over the one-year follow-up across most EQ-5D-5L dimensions with some uncertainties on why this was seen. More research is urgently needed to identify and understand why females and older patients have worse HRQoL and shows there is a need for more gender-and age-specific treatment in OAT programs. The wide variations in HRQoL support more emphasis on individualized treatment and personalized patient care, and the need for regular assessment of HRQoL in OAT programs. Our study is among the first to show that changes in HRQoL, including positive changes, are possible even several years after initiation of treatment. Future research should examine what causes these changes in HRQoL in long-term OAT patients.Additional file 1. OAT = opioid agonist therapy. Flow chart of study sample.Additional file 2. EQ-5D-5L, descriptive health profile at baseline (frequencies and proportions reported by dimensions and level). OAT = opioid agonist therapy, CI = confidence interval. * = total number of respondents. 8 patients who missed data on one dimension were included in the analysis. ** = long acting morphine sulphate and other opioid prescriptions.Additional file 3. Changes in EQ-5D-5L index value per patient from baseline to follow-up. 609 patients included at baseline, 245 patients at follow-up one year later.


FRMD7 for X-linked CMN. Four loci for autosomal dominant CMN, including NYS7 (OMIM 614826), have been mapped but the causative genes have yet to be identified. NYS7 was mapped to 1q32.1 based on independent genome-wide linkage scan on two large families with CMN. In this study, mutations in all known protein-coding genes, both intronic sequence with predicted effect and coding sequence, in the linkage interval were excluded by whole-genome sequencing. Then, long-read genome sequencing based on the Nanopore platform was performed with a sample from each of the two families. Two deletions with an overlapping region of 775,699 bp, located in a region without any known protein-coding genes, were identified in the two families in the linkage region. The two deletions as well as their breakpoints were confirmed by Sanger sequencing and co-segregated with CMN in the two families. The 775,699 bp deleted region contains uncharacterized non-protein-coding expressed sequences and pseudogenes but no protein-coding genes. However, Hi-C data predicted that the deletions span two topologically associated domains and probably lead to a change in the 3D genomic architecture. These results provide novel evidence of a strong association between structural variations in non-coding genomic regions and human hereditary diseases like CMN with a potential mechanism involving changes in 3D genome architecture, which provides clues regarding the molecular pathogenicity of CMN.Congenital motor nystagmus (CMN) is characterized by early-onset bilateral ocular oscillations without other ocular deficits. To date, mutations in only one gene have been identified to be responsible for CMN, i.e., The online version of this article (10.1007/s00439-020-02156-0) contains supplementary material, which is available to authorized users. Nystagmus is a condition characterized by involuntary oscillation of eyes. Infantile nystagmus, onset in the first 6 months of life, has been reported to occur in 1 of 821 live births  have been shown to be responsible for NYS1, which might account for 22–35% of CMN  , probably leading to a change in 3D genome architecture. These results provide novel evidence of a strong association between structural variants (SVs) in genomic regions devoid of protein-coding genes and human hereditary disease possibly by changing the 3D genome architecture.A total of forty-one individuals from two families with CMN were recruited for this study, including 25 affected individuals and 16 unaffected individuals as described in our previous studies Fig. . CMN in https://bio-bwa.sourceforge.net/). Variants, including single-nucleotide variants (SNVs), small insertions and deletions (InDels), and structural variations (SVs), were filtered using GATK (https://gatk.broadinstitute.org/hc/en-us) and annotated using ANNOVAR (https://annovar.openbioinformatics.org/en/latest/).Whole-genome sequencing was performed using genomic DNA from peripheral blood obtained from two affected members, i.e., III:1 from family A and IV:2 from family B  using the NimbleGen Human CGH 385 K Whole-Genome Array  with a capacity of 385,000 probes. In brief, genomic DNA from the affected member (IV:2) was considered the test DNA; while, genomic DNA from his unaffected sister (IV:1) was set as the reference DNA. The test sample (IV:2) was labeled with Cy3 dye and the reference sample (IV:1) was labeled with Cy5 dye. The labeled DNA was then hybridized with the NimbleGen CGH chip. Finally, the arrays were scanned using a GenePix4000B scanner  and analyzed using the NimbleScan 2.5.Whole-genome array-based comparative genomic hybridization (aCGH) was performed on DNA samples of two members, i.e., IV:1 and IV:2 from family A, by a commercial service provided by Macrogen (https://www.grandomics.com). The procedure for long-read genome sequencing has been previously described , and 1000 Genomes (https://phase3browser.1000genomes.org/index.html) databases.Long-read genome sequencing was performed on two affected individuals with CMN from the two families, i.e., II:9 from family A and III:7 from family B Fig. , by the Sanger sequencing was used to validate the potential pathogenic variants that were detected by the Oxford Nanopore platform and for segregation of candidate variants in the two families. Two pairs of primers were designed to validate the two SVs in the two families. The forward and reverse sequences of primers used for validation for the SV in family A were 5′-agaccatgggccaaagtgtt-3′ and 5′-agcatcagtagccgattcga-3′, respectively (M1-F and M1-R in Table S1). The forward and reverse sequences of primers used for validation for the SV in family B were 5′- gcttttctcctggtgctttca-3′ and 5′- accattgtactcccagcctg-3′, respectively (M2-F and M2-R in Table S1). An additional pair of primers (WT-F and WT-R in Table S1) was designed to validate hemizygosity of the region in the two families. The procedures that were used for amplification and sequencing of the targeted variants were previously described .To clarify the boundaries of the deletion, long-read sequencing by the Nanopore platform was performed on samples from two affected members from the two families. Two SVs within the linkage interval were detected: a deletion of 914,377 bp in family A and a deletion of 794,505 bp in family B. Neither of the two SVs was present in the DGV or 1000 Genome database. The exact deletions inside the linkage interval for the two families with CMN were confirmed by Sanger sequencing of the fragments containing the breakpoints, i.e., chr1: 198939222_199853599del (hg19 genome assembly) in family A and chr1: 199077900_199872405del (hg19 genome assembly) in family B . The overlapping deleted region was organized into two TADs based on both GM12878 (a human lymphoblastoid cell line) and fetal brain tissue  and five pseudogenes Fig. , Table 1LOC105371680) that shows low expression in normal human tissues, including the human brain, according to the NCBI database (https://www.ncbi.nlm.nih.gov/gene/?term=LOC105371680); while, the latter region is located at the boundary of the two TADs. Seven pathogenic/likely pathogenic copy number variations were reported according to the DECIPHER database (https://decipher.sanger.ac.uk), all of which involved not only the deletion regions in the present study but also multiple protein-coding genes .A total of 106 different SVs have been reported overlapping with the two SVs identified in our families according to the DGV database and two regions common to the SVs in our families were not covered by any of the 106 SVs, that is the 199,407,000–199,436,000 and 199,631,000–199,641,000 regions . The former region involves an ncRNA coding gene  . Another possibility is that the SVs could affect the expression of genes in a distal region  remained at the regions uncovered by the 106 reported SVs in the overlap of the two deletion regions. However, this ncRNA gene has a low expression in the human brain. Therefore, the association between the CMN phenotype and these ncRNA genes need to be investigated in additional families with CMN.Although six ncRNA genes are involved in the two deletion regions, three of them are locate at the overlapping region of the two deletions. Moreover, only the uncharacterized ncRNA coding gene Supplementary file2 Supplementary Figure 1. The log2 ratio plots of chromosome 1 in IV:2 from family A by aCGH. An approximately 0.89 Mb deletion was detected at chr1:197217711-198112096 based on GRCh37/hg19 (blue square). (TIF 1166 kb)Supplementary file3 Supplementary Figure 2. The SVs reported in DGV in the deletion interval of the two families. Two regions were not covered by any of these SVs, that is 199,407,000–199,436,000 and 199,631,000–199,641,000 regions, which have been indicated by red squares. (TIF 2295 kb)Supplementary file4 Supplementary Figure 3. SVs reported in DECIPHER database in the deletion interval of the two families. Seven pathogenic/likely pathogenic copy number variations were reported, all of which involved not only the deletion regions in the present study but also multiple protein-coding genes. (TIF 5634 kb)Below is the link to the electronic supplementary material.


Statins, including simvastatin (SMV), are commonly used for the control of hyperlipidaemia and have also proven therapeutic and preventative effects in cardiovascular diseases. Besides that, there is an emerging interest in their use as antineoplastic drugs as demonstrated by different studies showing their cytotoxic activity against different cancer cells. In this study, SMV-loaded emulsomes (SMV-EMLs) were formulated and evaluated for their cytotoxic activity in MCF-7 breast cancer cells. The emulsomes were prepared using a modified thin-film hydration technique. A Box–Behnken model was used to investigate the impact of formulation conditions on vesicle size and drug entrapment. The optimized formulation showed a spherical shape with a vesicle size of 112.42 ± 2.1 nm and an entrapment efficiency of 94.34 ± 1.11%. Assessment of cytotoxic activities indicated that the optimized SMV-EMLs formula exhibited significantly lower half maximal inhibitory concentration (IC50) against MCF-7 cells. Cell cycle analysis indicated the accumulation of cells in the G2-M phase as well as increased cell fraction in the pre-G1 phase, suggesting an enhancement of anti-apoptotic activity of SMV. The staining of cells with Annex V revealed an increase in early and late apoptosis, in line with the increased cellular content of caspase-3 and Bax. In addition, the mitochondrial membrane potential (MMP) was significantly decreased. In conclusion, SMV-EMLs demonstrated superior cell death-inducing activity against MCF-7 cells compared to pure SMV. This is mediated, at least in part, by enhanced pro-apoptotic activity and MMP modulation of SMV. Breast cancer is the most common type of cancer in women, which is frequently detected as an abnormal lump in the breast tissue ,2. BreasSMV is on the list of essential medicines published by the World Health Organization (WHO) as the most effective and safest medicines required for a health system . Lately,Liposomes are carrier systems that were first described more than 50 years ago . These aThe aim of this work was to investigate the ability of EMLs to improve the cytotoxic and apoptotic efficacy of SMV in human breast cancer (MCF-7) cells. This specific cell line was selected because it represents an established in vitro experimental model in drug discovery processes in the field of oncology to study the toxic potential of SMV alone or in combination with other antineoplastic drugs ,29,30,31® 90 H  was a gift received from Lipoid GmbH .All chemicals were of analytical grade and purchased from Sigma-Aldrich Corporate  or Thermo Fisher Scientific Inc.  unless specified otherwise. SMV was supplied as a kind gift from Jamjoom Pharma , while Phospholiponw/w, X1), Phospholipon® 90 H concentration , and ultrasonication time , while vesicle size  and entrapment efficiency  were studied as responses. The coded levels nominated as  for each factor and their corresponding actual values are shown in A Box–Behnken experimental design was utilized for SMV-EMLs formulation using Design-Expert software . The independent variables were SMV concentration . Analysis of variance (ANOVA) was used to statistically analyze the responses, and the statistical significance level was set at 95%. To explore the interaction between the studied variables, three-dimensional surface plots were produced. Statistical analysis was performed to select the best-fitting model for each response parameter based on the predicted and adjusted correlation coefficient , and tripalmitin (2% w/v) in 20 mL of chloroform/methanol mixture  in a round-bottom flask. A rotavapor at 40 °C was used for the evaporation of the solvent under reduced pressure. To ensure the removal of organic solvent, the deposited films were kept in a vacuum oven overnight for complete drying  assay as previously described [2. On the day of the experiment, MCF-7 cells were left untreated (control) or treated with Placebo-EMLs, pure SMV (SMV-raw), or SMV-loaded EMLs (SMV-EMLs) for 24 h. Next, the medium was removed from each well, and the MTT solution (1 mg/mL in medium) was added to the cell cultures and incubated for two h at 37 °C. The formed crystals were melted by using 200 μL of dimethyl sulfoxide (DMSO), and the absorbance at 569 nm was read with a Spark® multimode microplate reader . The IC50 for Placebo-EMLs, pure SMV (SMV-raw), or SMV-loaded EMLs (SMV-EMLs) was calculated based on the curves obtained measuring the variation of cell viability (%) as a function of increasing concentrations  of SMV-raw or SMV-EMLs, the IC50 of SMV-raw. Data are the mean of 3 independent experiments.The IC50 values of MCF-7 breast cancer cells plated in 96-well plates , were left untreated or treated and incubated for 24 h. CycleTEST™ PLUS DNA Reagent Kit  was used to analyze the cell cycle according to the manufacturer’s instructions. Control cells  with known DNA content provided a reference point for determining the DNA index for a test sample. Data analysis was carried out by using MultiCycle AV software .The study of the cell cycle as well as apoptosis and necrosis in untreated MCF-7 cells or treated with Placebo-EMLs, SMV-raw, or SMV-EMLs for 24 h was performed by flow cytometry . SMV was used at a concentration of 0.1 µM. The procedure followed by studying the cell cycle is the same method previously described elsewhere . MCF-7 cThe Annexin V-FITC Apoptosis Kit , allowing for the detection of different stages of apoptosis and differentiates apoptosis from necrosis (performing both Annexin V-FITC and propidium iodide (PI) staining) was used to study cell death following the instructions provided by the manufacturer. Since PI is able to enter into damaged and dead cells only, its use allows to distinguish between apoptosis and necrosis.5 cells/well) by monitoring the changes in MMP using a MitoProbe™ TMRM Assay Kit for Flow Cytometry  according to the manufacturer’s instructions. SMV was used at a concentration of 0.1 µM. This assay is based on the use of a cell-permeant, cationic, tetramethylrhodamine methyl ester (TMRM) probe that is readily sequestered by active mitochondria, thereby emitting a red-orange fluorescent signal. When apoptosis is induced, the MMP is depolarized, and the TMRM signal diminishes. The cells were kept in the dark with a TMRM reagent solution for the suggested time, washed with PBS to remove any residual probe, added of live-cell imaging buffer, and analyzed by using a flow cytometer .Mitochondrial function, one of the major determinant and indicator of cell fate, was assessed in untreated or treated  MCF-7 cells plated in 96-well plates (1 × 104 cells/well) according to the protocol supplied by the manufacturer. SMV was used at a concentration of 0.1 μM. Following treatment for 24 h, the MCF-7 cells were washed, lysed by using a cell extraction buffer, and the obtained lysates were used for assaying caspase-3 activity by reading the absorbance at 405 nm with a Spark® multimode microplate reader .Caspase-3 Assay Kit  was employed to measure caspase-3 activity in untreated or treated  MCF-7 cells plated in 96-well plates (5 × 10® multimode microplate reader (Tecan Group Ltd.). A correction between 570 and 590 nm was applied.The quantitative determination of Bax protein in the MCF-7 lysates obtained by MCF-7 cells subjected to the different treatments  was carried out by using Human Bax ELISA kit  according to the manufacturer’s instructions. SMV was used at a concentration of 0.1 µM. Briefly, the different cell lysates were added to the microtiter plate containing a monoclonal antibody for Bax protein. After a short incubation, the excess sample or standard (a recombinant human Bax-α standard) was washed out, and a biotinylated monoclonal antibody for Bax, able to bind to the Bax captured on the plate, was added. After a short incubation followed by a washing step, streptavidin conjugated to horseradish peroxidase was added. The enzyme reaction was blocked by the addition of a stop solution and the optical density read at 450 nm was measured by using a Sparkp < 0.05 were considered statistically significant. Each set of experiments was performed at least in triplicate and is reported as means ± SD.The software selected to perform the statistical analysis was Graphpad Prism . One-way or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used for multiple comparisons. Only values of 2 and the lowest predicted residual error sum of squares (PRESS)  .2 were in reasonable agreement, indicating the validity of the model. Adequate signal to noise ratio is depicted by adequate precision greater than 4 for both responses; this ratio confirms the suitability of the selected models to navigate the experimental design space. Moreover, the predicted and adjusted RDiagnostic plots for both responses were produced to evaluate the goodness of fit of the selected models and confirm their validity . Figure Also, the predicted vs. actual values of both responses’ plots A2,B2 demp = 0.0054). Non-significant lack of fit, depicted by the lack of fit F-value of 3.34 (p = 0.1374), ensured data fitting to the selected model. Equation (2), representing the quadratic model, was generated in terms of coded factor:SMV-EMLs exhibited nano-sized vesicles with an average size ranging from 71 ± 0.46 to 172 ± 1.45 . The rel1, X2, and X3), corresponding to the investigated variables, have a significant effect on vesicle size at a 95% level of significance. The quadratic term corresponding to the ultrasonication time (X32) in addition to the interaction terms X1X3 and X2X3 corresponding to the interaction between ultrasonication time and either SMV concentration or Phospholipon® 90 H concentration were also found to be significant at the same level. ANOVA results revealed that all the linear terms . The observed trend is confirmed by the positive coefficients of both X1 and X2 in the coded equation. The effect of phospholipid concentration was more pronounced than that of the drug, as evidenced by the higher coefficient of its corresponding term. On the other hand, the vesicle size significantly decreased with increasing ultrasonication time (p < 0.0001). This reduction could be ascribed to the main principle underlying the sonication process. It was evident that the vesicle size significantly increases with increasing both SMV and Phospholiponp < 0.0001). Non-significant lack of fit, depicted by the lack of fit F-value of 2.44 (p = 0.2024), ensured data fitting to the selected model. Equation (3), representing the linear model, was generated in terms of coded factor:The prepared SMV-EMLs showed marked variation in drug entrapment with entrapment efficiency ranging from 76.9 ± 0.65 to 96.7 ± 0.83. ANOVA for the entrapment efficiency confirmed fitting of the data to the linear model, as evidenced by its F-value of 130.28 (1 and X2 corresponding to both SMV and Phospholipon® 90 H concentrations displayed a significant positive impact on the entrapment efficiency as proved by their positive coefficients (p < 0.0001). ANOVA results revealed that both terms XThe vesicle size and entrapment efficiency constraints were used for the prediction of the optimized levels of the variables utilizing a numerical optimization approach. The desirability function was computed as 0.732. The optimized formulation was prepared and assessed. The percentage error between the predicted and observed responses was relatively low, indicating that the optimization technique used was valid. The PDI of the optimized formulation was found to be 0.221 ± 0.012, while the zeta potential was equal to 10.17 ± 1.23 mV. The stability test revealed a statistically non-significant difference in the entrapment efficiency %, zeta potential, or particle size. All formulations revealed good dispersion stability. The optimized formulation was subjected to further investigations. The variables levels predicted, and observed responses for the optimized formulation are depicted in Representative TEM micrograph of the optimized SMV-EMLs is shown in The in vitro dissolution of SMV from the optimized EMLs was studied. The drug release profile is shown in A controlled and gradual release profile was observed for the prepared SMV-EMLs. Around 80% of drug release was achieved within 12 h. SMV released from EMLs revealed slower release when compared to SMV-raw. The drug release was almost complete by 24 h. The cumulative amount released could be regarded as satisfactory, considering the poor solubility of SMV. p < 0.05 vs. all)  on MCF-7 cells, we carried out the MTT assay. The lowest IC50 value, thus the highest toxic potential, was observed in cells treated for 24 h with SMV-EMLs  .SMV-raw exhibited a weaker activity compared to SMV-EMLs, with an IC50 value of about 5-fold higher (IC50 = 41.75 μM) than that observed for the drug encapsulated into emulsomes. As expected, the highest IC50 was observed for the treatment with Placebo-EMLs, then in the absence of drug (IC50 = 78.29 μM). From this set of data, we were able to select the SMV-raw concentration (0.1 µM) that produced a negligible decrease in cell viability in MCF-7 cells and compare it with the same concentration of optimized SMV-EMLs, which exhibits nano-size and enhanced drug entrapment with gradual and complete in vitro release, inquiring into a possible enhancement for the antineoplastic effects of the drug . The selp < 0.05) only in the case of S and pre-G1 phases with a significant reduction of the percent of cells in the S phase (p < 0.05), with a significant and biologically-relevant accumulation of cells in the G2/M and pre-G1 phases combined with a significant reduction of cells in the S phase  was observed when considering the sum of apoptotic + necrotic cell populations. As expected, the treatment with optimized SMV-EMLs led to a significant increase in the percentage of cells in apoptosis + necrosis , early and late stages of apoptosis, and necrosis compared to untreated cells as well as to Placebo-EMLs and SMV-raw-treated MCF-7 cells (p < 0.05), underlining the highly relevant pro-apoptotic activity of SMV-EMLs. It is clear that Placebo-EMLs and SMV-raw treatments did not lead to any significant changes in the apoptotic stages and necrosis compared to untreated cells A–C,E. ThNo significant difference in MMP was observed when considering untreated, Placebo-EMLs, and SMV-raw-treated MCF-7 cells . p < 0.05) reduction of MMP compared to all the other treatments is represented by optimized SMV-EMLs.The only treatment able to give a significant (In line with the results shown in p < 0.05), while the highest content of caspase-3 was detected in cells stimulated with optimized SMV-EMLs (22.5 ± 2.4 pg/mg protein), that was significantly higher compared to untreated cells as well as to Placebo-EMLs and SMV-raw treatments .Exposure of the cells to SMV-raw gave a content of caspase-3 about 2.5-fold higher than that of control cells (5.23 ± 1.32 pg/mg protein) (p < 0.05).p < 0.05) was observed.Once again, the treatment giving the more evident changes was represented by optimized SMV-EMLs, where an increase of about ~5-fold higher than that of control cells (p < 0.0001). This reduction could be ascribed to the main principle underlying the sonication process, generating cavitation bubbles in the EMLs dispersion by the ultrasound mechanical waves. Bubbles having a size close to the resonant size for the employed frequency starts to oscillate non-linearly and collapse, resulting in bubble implosion that generates extremely high temperatures, high pressures, and shock waves. Thus, the ultrasonic generated high energy result in size reduction [Statins, including SMV, are poorly soluble drugs and drug delivery approaches such as emulsomes  represeneduction ,42. Accoeduction . SMV, beeduction ,45. The eduction . The zetSMV is currently used and approved to lower cholesterol production and reduce dyslipidemia associated complications, because of its ability to reduce LDL by inhibiting the hydroxymethylglutaryl coenzyme A reductase, and then the hepatic synthesis of cholesterol . This drBased on the above evidence, we performed in vitro experiments in which the ability of EMLs to enhance the toxic potential of SMV towards MCF-7 cells, representing a well-known experimental model in cancer research , was tesThe present study clearly shows the enhanced cytotoxic activity exerted by the optimized SMV-EMLs formula on the well-characterized MCF-7 cancer cell model ,30,31,53In the present study, a Box–Behnken design was successfully applied for the formulation and optimization of SMV-EMLs. The prepared EMLs exhibited nano-size and enhanced drug entrapment exceeding 80%. The optimized EMLs formulation with minimized vesicle size and maximized drug entrapment exhibited spherical shape with gradual and complete in vitro release. The in vitro drug release indicated the ability of the EMLs to enhance the dissolution of the poorly soluble drug SMV. By performing in vitro experiments with MCF-7 cancer cells, it was possible to show that drug loading of EMLs (SMV-EMLs) significantly improved the anti-proliferative activity and enhanced the toxic and pro-apoptotic effects of SMV towards cancer cells as demonstrated by the lower IC50. Additionally, SMV-EMLs containing a sub-toxic concentration of drug led to the inhibition of cell proliferation, the increase of apoptotic and necrotic cell populations, the decrease of the mitochondrial membrane potential, the increase of the intracellular concentration of caspase-3, and the increased intracellular levels of Bax protein compared to the treatment with SMV in the absence of EMLs. Our data obtained by using an optimized SMV-EMLs formula suggest that SMV-EMLs might represent a novel path for the development of a specific and more effective treatment of breast cancer.